Polyomavirus BK (BKV) is now the commonest cause of viral infection after kidney transplantation. BK viruria, viremia and nephropathy (BKVN) occur in respectively 20-60%, 5-20%, and 1-10% of kidney transplant recipients. T-cell mediated rejection (TCMR) complicates the course of up to 30% cases of BKVN. The diagnosis of TCMR in patients passing through the inflammatory phase of BKVN is a problematic area. Both rejection and BKVN are characterized by T-cell rich infiltrates that look alike. Light microscopy cannot determine whether viral or HLA antigens are being primarily targeted by these cells in any given biopsy. There is an unmet medical need to develop a laboratory test that can diagnose TCMR in the face of viral nephropathy. It is our contention that sequencing of the ?? T-cell receptor (TCR) can provide a basis for the development of such a test, since the TCR sequence can provide information about the nature of the antigen to which a particular T-cell is responding. Accordingly, we have devised a research plan comprised of two Specific Aims that can be summarized as follows. In Specific Aim 1, we will use next generation sequencing (NGS) of the T-cell receptor to characterize the repertoire of circulating T-cells used by kidney patient to respond to donor HLA or BKV antigens. In Specific Aim 2, we will utilize these anti-donor and anti-BKV TCR signatures for distinguishing between TCMR and BKVN in allograft biopsy tissue. T-cell clones that expand at least 5 fold in response to alloantigen or BKV antigen stimulation will be tabulated. The mean number and cumulative frequency of donor and BKV reactive clones will then be compared in biopsies taken at the time of clinical stability, TCMR or BKVN. The clonal frequencies that best discriminate between these 3 different clinical scenarios will be determined by receiver operating curves. Patients with BKVN will be divided into categories, namely those with and without concurrent NGS criteria of TCMR. The graft function in these two groups of patients will be followed without any anti-rejection therapy. Worse outcome in patients with expansion of alloreactive T-cell clones will further validate TCR NGS as a suitable tool for the diagnosis of TCMR in the setting of BKVN. Relevance to Public Health: In the short term, the proposed studies are important in that they will allow development of a test that can detect the onset of T-cell mediated rejection in patients with BKVN. A positive test would identify a subset of patients who would need accentuation of immunosuppression. Conversely, a negative test would rule out TCMR and enable reduction of immunosuppression, restoration of cell mediated immunity, and resolution of BKV mediated tissue injury. Thus, following appropriate validation, implementation of T-cell repertoire testing in the transplant clinic will permit alloimmune monitoring and personalized anti- rejection treatment in patients with BKV nephropathy. In the long run, the work proposed will help usher in an era of personalized medicine for transplant patients. Potential applications of NGS-based tracking of individual T-cell clones would include the ability to: (i) predict the development of acute and chronic rejection, (ii) identify patients at very low risk for rejection and suitable for entry in toleranc induction protocols), (iii) stratify patients by risk for developing viral infection, (iv) predict linical outcome, and (v) determine the endpoint for antiviral therapy. The technology to be developed will be relevant to microbiologists studying bacterial, fungal and other viral diseases, and to immunologists interested in caring for patients with non-viral infectious disease, autoimmune disorders, and primary or acquired immunodeficiency.